Among pharmaceutical technologies, PEGylation of peptides and proteins for the purpose of treatment is the most effective technology. PEGylation of peptides and proteins increases molecular weight thereof, protein degradation site defense and immunogenicity site defense, which consequently increases half-life of in vivo medications and reduce immunogenicity of peptides and proteins. Therefore, PEGylation technology has an effect of increasing treatment effect by solving problems of original medications, and due to such strength, serves an important role in increasing effects of PEGylated peptide and protein medication delivery system.
Also, peptides and proteins increase treatment effect by covalently bonding with polyethylene glycol (PEG). Such technology increases molecular weight, defense of a metabolism site and inhibition of an immunogenicity site, increasing in vivo half-life and stability and reducing immunogenicity. Furthermore, kidney excretion of peptides and proteins bound with PEG is reduced due to the increase of molecular weights of peptides and proteins by PEG, so that PEGylation has advantages of increasing effects in both pharmacokinetically and pharmacodynamically.
PEGylation reacting sites of peptides and proteins are randomly dispersed and are occasionally close to bioactive sites. However, traditional PEGylation employs nonspecific PEGylation methods that do not consider PEG reacting site, number of PEG bonds and biological activity. However, such a nonspecific PEGylation method reduces treatment effects by bringing insufficient conformation by producing various branched type PEG-bonded isomers that have different physiochemical, biological and pharmacokinetic characteristics. Specific PEGylation methods have been studied to solve such problems, and recently the specific PEGylation methods are rapidly developing to become a method of maximizing medications' treatment effects as genetic engineering technology and selective functional group introducing technology are quickly developing. In a related art, a study of selectively binding PEG into N-terminal site after removing a reaction site by substituting primary amine site with different amino acid using genetic engineering method for granulocyte stimulating factor (G-CSF) and tumor necrosis factor receptor has been conducted previously.
Also, studies using a technology that selectively PEGylates substituent after having introduced a specific substituent using genetic engineering methods and substitution technology for medications such as staphylokinase, interferon a-2, antibody single chain fragment variable (ScFv), have been conducted.
Exendin-4 is a polypeptide substance and is the first incretin analogue, a diabetes medication prepared by synthesizing exendin-4, a salivary substance of Gila monster. Exendin-4 is different from exendin-3 for only #2 and #3 sites, is known to have a longer half-life than glucagon like peptide-1 (GLP-1) which is a diabetes medication having a half-life shorter than two minutes for DPP-IV, an enzyme that is resistant for directly degrading incretin enzyme that is produced in mammals' stomachs after ingestion by DPP-IV (dipeptidyl peptidase-4) to serve beneficial roles of promoting insulin secretion and lowering blood sugar level, and also, it shows 2-4 hours of half-life in vivo experiment, and it has been confirmed that it can reach enough blood concentration with 2-3 times of intraperitoneal injection per day.
Also, exendin-4 is known to control gastrointestinal tracts' motility, reduces food intake and suppresses blood plasma glucagon, and recently PLGA microsphere type synthetic exendin-4 (product name: Byetta) has been authorized by US FDA and is about to be released. However, since this Byetta LAR product has complicated preparation process and is short in vivo half-life for exendin-4, which is about 4-6 hours, frequent administration of high dose exendin-4 is required, and the problem of medication release control based on quick excretion due to the low molecular weight of lower than 4200, and problems such as immunogenicity still exist.
Therefore, while studying a method to reduce administration frequency of exendin-4 and solve the low molecular weight problem of exendin-4, the inventors have completed the present invention after having confirmed the fact that it is possible to increase the production yield of PEGylated exendin-4 and treatment effect of medications by performing selective PEGylation via insertion of cysteine (Cys) amino acid into the site (#40 site) next to #39 site of C-terminal of exendin-4.